1, 3, 4-oxadiazol-2-ones as ppar delta modulators and their use thereof

ABSTRACT

The present invention is directed to 1,3,4-oxadiazalones, compounds of formula I and their pharmaceutically acceptable salts stereoisomers, tautomers, or solvates thereof. Novel compounds include those of formula I, in which radicals are as defined herein. 
     
       
         
         
             
             
         
       
     
     The compounds of this invention are modulators of PPARdelta and therefore useful as pharmaceutical agents, especially for the treatment of demyelinating diseases and disorders of fatty acid metabolism and glucose utilization.

FIELD OF THE INVENTION

This invention relates to novel compounds and pharmaceuticalformulations that act as selective PPARdelta ligand receptor binders,which are useful in modulating PPARdelta receptors for the treatment ofdiseases mediated by nuclear hormone receptors. The PPARdelta ligandreceptor binders of this invention are useful as agonists or antagonistsof the PPARdelta receptor.

BACKGROUND OF THE INVENTION

The peroxisome proliferator-activated receptors (PPARs) comprise asubfamily of the nuclear receptor superfamily. Four closely relatedisoforms have been identified and cloned and are commonly known asPPARalpha, PPARgamma-1, PPARgamma-2 and PPARdelta. Each receptor subtypehas a signature DNA binding domain (DBD) and a ligand-binding domain(LBD), both being necessary for ligand activated gene expression. PPARsbind as heterodimers with a retinoid X receptor. See J. Berger and D. E.Miller, Annu. Rev. Med., 2002, 53, 409-435.

PPARdelta (also known as PPARbeta) is expressed in a broad range ofmammalian tissue, but little information regarding its biologicalfunctions or the full array of genes regulated by the receptor have beenelucidated. However, it has recently been found that agonists may beuseful to treat conditions such as dyslipedemia and certaindermatological conditions, while antagonists may be useful to treatosteoporosis or colorectal cancer (D. Sternbach, in Annual Reports inMedicinal Chemistry, Volume 38, A. M. Doherty, ed., Elsevier AcademicPress, 2003 pp. 71-80).

PPARdelta appears to be significantly expressed in the CNS; however muchof its function there still remains undiscovered. Of singular interesthowever, is the discovery that PPARdelta was expressed in rodentoligodendrocytes, the major lipid producing cells of the CNS (J.Granneman, et al., J. Neurosci. Res., 1998, 51, 563-573). Moreover, itwas also found that a PPARdelta selective agonist was found tosignificantly increase oligodendroglial myelin gene expression andmyelin sheath diameter in mouse cultures (I. Saluja et al., Glia, 2001,33, 194-204). Thus, PPARdelta activators may be of use for the treatmentof demyelinating and dysmyelinating diseases.

Demyelinating conditions are manifested in loss of myelin—the multipledense layers of lipids and protein which cover many nerve fibers. Theselayers are provided by oligodendroglia in the central nervous system(CNS), and Schwann cells in the peripheral nervous system (PNS). Inpatients with demyelinating conditions, demyelination may beirreversible; it is usually accompanied or followed by axonaldegeneration, and often by cellular degeneration. Demyelination canoccur as a result of neuronal damage or damage to the myelinitself—whether due to aberrant immune responses, local injury, ischemia,metabolic disorders, toxic agents, or viral infections (Prineas andMcDonald, Demyelinating Diseases. In Greenfield's Neuropathology,6.sup.th ed. (Edward Arnold: New York, 1997) 813-811, Beers and Berkow,eds., The Merck Manual of Diagnosis and Therapy, 17.sup.th ed.(Whitehouse Station, N.J.: Merck Research Laboratories, 1999) 1299,1437, 1473-76, 1483).

Central demyelination (demyelination of the CNS) occurs in severalconditions, often of uncertain etiology, that have come to be known asthe primary demyelinating diseases. Of these, multiple sclerosis (MS) isthe most prevalent. Other primary demyelinating diseases includeadrenoleukodystrophy (ALD), adrenomyeloneuropathy, AIDS-vacuolarmyelopathy, HTLV-associated myelopathy, Leber's hereditary opticatrophy, progressive multifocal leukoencephalopathy (PML), subacutesclerosing panencephalitis, Guillian-Barre syndrome and tropical spasticparaparesis. In addition, there are acute conditions in whichdemyelination can occur in the CNS, e.g., acute disseminatedencephalomyelitis (ADEM) and acute viral encephalitis. Furthermore,acute transverse myelitis, a syndrome in which an acute spinal cordtransection of unknown cause affects both gray and white matter in oneor more adjacent thoracic segments, can also result in demyelination.Also, disorders in which myelin forming glial cells are damagedincluding spinal cord injuries, neuropathies and nerve injury.

Selective PPARdelta modulators may also be useful for treating orpreventing other disease states see, for example, Joel Berger et al.,Annu. Rev. Med. 2002, 53, 409-435; Timothy Wilson et al. J. Med. Chem.,2000, Vol. 43, No. 4, 527-550; Steven Kliewer et al., Recent Prog HormRes. 2001; 56: 239-63; Jean-Charles Fruchart, Bart Staels and PatrickDuriez: PPARS, Metabolic Disease and Arteriosclerosis, PharmacologicalResearch, Vol. 44, No. 5, 345-52; 2001; Sander Kersten, BeatriceDesvergne & Walter Wahli: Roles of PPARs in health and disease, Nature,vol 405, 25 may 2000; 421-4; Ines Pineda Torra, Giulia Chinetti,Caroline Duval, Jean-Charles Fruchart and Bart Staels: Peroxisomeproliferator-activated receptors: from transcriptional control toclinical practice, Curr Opin Lipidol 12: 2001, 245-254).

Compounds acting as PPARdelta modulators may be particularly suitablefor the treatment and/or prevention of disorders of fatty acidmetabolism and glucose utilization disorders in which insulin resistanceis involved.

Diabetes mellitus, especially type 2 diabetes, including the preventionof the sequelae associated therewith. Particular aspects in thisconnection are hyperglycemia, improvement in insulin resistance,improvement in glucose tolerance, protection of the pancreatic B cells,prevention of macro- and microvascular disorders.

Dyslipidemias and their sequelae such as, for example, atherosclerosis,coronary heart disease, cerebrovascular disorders etc, especially those(but not restricted thereto) which are characterized by one or more ofthe following factors: high plasma triglyceride concentrations, highpostprandial plasma triglyceride concentrations, low HDL cholesterolconcentrations, low ApoA lipoprotein concentrations, high LDLcholesterol concentrations, small dense LDL cholesterol particles, highApoB lipoprotein concentrations.

Various other conditions which may be associated with the metabolicsyndrome, such as: obesity (excess weight), including central obesity,thromboses, hypercoagulable and prothrombotic states (arterial andvenous), high blood pressure, heart failure such as, for example (butnot restricted thereto), following myocardial infarction, hypertensiveheart disease or cardiomyopathy.

Other disorders or conditions in which inflammatory reactions or celldifferentiation, may for example be involved are: atherosclerosis suchas, for example (but not restricted thereto), coronary sclerosisincluding angina pectoris or myocardial infarction, stroke, vascularrestenosis or reocclusion, chronic inflammatory bowel diseases, such as,for example, Crohn's disease and ulcerative colitis, pancreatitis, otherinflammatory states, retinopathy, adipose cell tumors, lipomatouscarcinomas such as, for example, liposarcomas, solid tumors andneoplasms such as, for example (but not restricted thereto), carcinomasof the gastrointestinal tract, of the liver, of the biliary tract and ofthe pancreas, endocrine tumors, carcinomas of the lungs, of the kidneysand the urinary tract, of the genital tract, prostate carcinomas etc.,acute and chronic myeloproliferative disorders and lymphomasangiogenesis, neurodegenerative disorders,, Alzheimer's disease,Parkinson's disease, erythemato-squamous dermatoses such as, forexample, psoriasis, acne vulgaris.

Other skin disorders and dermatological conditions modulated byPPARdelta: eczemas and neurodermitis, dermatitis such as, for example,seborrheic dermatitis or photodermatitis, keratitis and keratoses suchas, for example, seborrheic keratoses, senile keratoses, actinickeratosis, photo-induced keratoses or keratosis follicularis keloids andkeloid prophylaxis, warts, including condylomata or condylomataacuminata, human papilloma viral (HPV) infections such as, for example,venereal papillomata, viral warts such as, for example, molluscumcontagiosum, leukoplakiapapular dermatoses such as, for example, Lichenplanus, skin cancer such as, for example, basal-cell carcinomas,melanomas or cutaneous T-cell lymphomas, localized benign epidermaltumors such as, for example, keratoderma, epidermal naevi andchilblains.

Various other conditions potentially modulated by PPARdelta includingsyndrome X, polycystic ovary syndrome (PCOS), asthma osteoarthritis,lupus erythematosus (LE) or inflammatory rheumatic disorders such as,for example, rheumatoid arthritis, vasculitis, wasting (cachexia), goutischemia/reperfusion syndrome and acute respiratory distress syndrome(ARDS).

SUMMARY OF THE INVENTION

The present invention is directed to compound of formula I.

whereinARYL is phenyl or pyridinyl, wherein said phenyl or pyridinyl isoptionally substituted with one or more substituents selected from thegroup consisting of halogen, C₁₋₆alkyl, C₂₋₆alkenyl, C₁₋₆alkoxy,C₁₋₆perfluoroalkyl; C₁₋₆alkylthio, hydroxy, hydroxyC₁₋₆alkyl,C₁₋₄acyloxy, nitro, cyano, C₁₋₆alkylsulfonyl, amino, C₁₋₆alkylamino andC₁₋₆alkoxycarbonyl;Z is —O(CH₂)_(n)—, —SO₂(CH₂)_(n)—, —(CH₂)_(n)—Y—(CH₂)_(n)—,—(CH₂)_(n)—CO—, —O(CH₂)_(n)—CO— or —(CH₂)_(n)—Y—(CH₂)_(n)—CO— wherein Yis NR₃, O or S and R₃ is selected from the group consisting of H,C₁₋₆alkyl C₃₋₈cycloalkyl, C₁₋₆alkylC₃₋₈cycloalkyl and benzyl and n isindependently an integer from 1 to 5;X is NR₃, O or S wherein R₃ is as defined above;R₁ is H, halogen, C₁₋₆alkyl, C₁₋₆alkoxy, C₁₋₆perfluoroalkyl;hydroxyC₁₋₆alkyl, nitro, cyano, and C₁₋₆alkylamino; andR₂ is substituted or unsubstituted phenyl, pyridinyl or thienyl whereinthe substituents are selected from the group consisting of halogen,C₁₋₆alkyl, C₂₋₆alkenyl, C₁₋₆alkoxy, C₁₋₆perfluoroalkyl, C₁₋₆alkylthio,hydroxy, hydroxyC₁₋₆alkyl, C₁₋₄acyloxy, nitro, cyano, C₁₋₆alkylsulfonyl,amino, C₁₋₆alkylamino and C₁₋₆alkoxycarbonyl;with the proviso that when Z is —O(CH₂)_(n)— or —SO₂(CH₂)_(n)—, and ARYLis phenyl then R₂ is other than phenyl;or a stereoisomer, a tautomer or a solvate thereof or a pharmaceuticallyacceptable salt thereof.

The present invention is also directed to pharmaceutical compositions offormula I, and methods of using said compounds and compositions formodulating PPARdelta in a subject in need of such modulation byadministering a compound which preferentially modulates the activity ofPPARdelta.

Another aspect of this invention is disclosed a method of treating adisease in a mammal wherein the disease is capable of being, modulatedby PPARdelta ligand binding activity, which comprises administering tosaid mammal having said disease a therapeutically effective amount of acompound of formula I.

whereinARYL is phenyl or pyridinyl, wherein said phenyl or pyridinyl isoptionally substituted with one or more substituents selected from thegroup consisting of halogen, C₁₋₆alkyl, C₂₋₆alkenyl, C₁₋₆alkoxy,C₁₋₆perfluoroalkyl; C₁₋₆alkylthio, hydroxy, hydroxyC₁₋₆alkyl,C₁₋₄acyloxy, nitro, cyano, C₁₋₆alkylsulfonyl, amino, C₁₋₆alkylamino andC₁₋₆alkoxycarbonyl;Z is —O(CH₂)_(n)—, —SO₂(CH₂)_(n)—, —(CH₂)_(n)—Y—(CH₂)_(n)—,—(CH₂)_(n)—CO—, —O(CH₂)_(n)—CO— or —(CH₂)_(n)—Y—(CH₂)_(n)—CO— wherein Yis NR₃, O or S and R₃ is selected from the group consisting of H,C₁₋₆alkyl C₃₋₈cycloalkyl, C₁₋₆alkylC₃₋₈cycloalkyl and benzyl and n isindependently an integer from 1 to 5;X is NR₃, O or S wherein R₃ is as defined above;R₁ is H, halogen, C₁₋₆alkyl, C₁₋₆alkoxy, C₁₋₆perfluoroalkyl;hydroxyC₁₋₆alkyl, nitro, cyano, and C₁₋₆alkylamino; andR₂ is substituted or unsubstituted phenyl, pyridinyl or thienyl whereinthe substituents are selected from the group consisting of halogen,C₁₋₆alkyl, C₂₋₆alkenyl, C₁₋₆alkoxy, C₁₋₆perfluoroalkyl, C₁₋₆alkylthio,hydroxy, hydroxyC₁₋₆alkyl, C₁₋₄acyloxy, nitro, cyano, C₁₋₆alkylsulfonyl,amino, C₁₋₆alkylamino and C₁₋₆alkoxycarbonyl; or a stereoisomer, atautomer or a solvate thereof or a pharmaceutically acceptable saltthereof.

DETAILED DESCRIPTION OF THE INVENTION

The terms as used herein have the following meanings:

As used herein, the expression “C₁₋₆alkyl” includes methyl and ethylgroups, and straight-chained or branched propyl, butyl, pentyl and hexylgroups. Particular alkyl groups are methyl, ethyl, n-propyl, isopropyland tert-butyl. Derived expressions such as “C₁₋₆alkoxy”,“C₁₋₆alkoxyC₁₋₆alkyl”, “hydroxyC₁₋₆alkyl”, “C₁₋₆alkylcarbonyl”,“C₁₋₆alkoxycarbonylC₁₋₆alkyl”, “C₁₋₆alkoxycarbonyl”, “aminoC₁₋₆alkyl”,“C₁₋₆alkylcarbamoylC₁₋₆alkyl”, “C₁₋₆dialkylcarbamoylC₁₋₆alkyl” “mono- ordi-C₁₋₆alkylaminoC₁₋₆alkyl”, aminoC₁₋₆alkylcarbonyl”,“diphenylC₁₋₆alkyl”, “arylC₁₋₆alkyl”, “arylcarbonylC₁₋₆alkyl” and“aryloxyC₁₋₆alkyl” are to be construed accordingly.

As used herein, the expression “C₂₋₆alkenyl” includes ethenyl andstraight-chained or branched propenyl, butenyl, pentenyl and hexenylgroups. Similarly, the expression “C₂₋₆alkynyl” includes ethynyl andpropynyl, and straight-chained or branched butynyl, pentynyl and hexynylgroups.

As used herein, the term “C₁₋₄acyloxy” denotes an acyl radical attachedto an oxygen atom, some examples include but not limited to acetyloxy,propionyloxy, butanoyloxy, iso-butanoyloxy, sec-butanoyloxy,t-butanoyloxy and the like.

As used herein “aryl” represents a carbocyclic aromatic ring system suchas phenyl, biphenyl, naphthyl, anthracenyl, phenanthrenyl, fluorenyl,indenyl, pentalenyl, azulenyl, biphenylenyl and the like. Aryl is alsointended to include the partially hydrogenated derivatives of thecarbocyclic aromatic systems enumerated above. Non-limiting examples ofsuch partially hydrogenated derivatives are 1,2,3,4-tetrahydronaphthyl,1,4-dihydronaphthyl and the like.

As used herein “aryloxy” represents a group—O-aryl wherein aryl is asdefined above.

As used herein “heteroaryl” (on its own or in any combination, such as“heteroaryloxy”, or “heteroaryl alkyl”)—a 5-10 membered aromatic ringsystem in which one or more rings contain one or more heteroatomsselected from the group consisting of N, O or S, such as, but notlimited, to pyrrole, pyrazole, furan, thiophene, quinoline,isoquinoline, quinazolinyl, pyridine, pyrimidine, oxazole, thiazole,thiadiazole, tetrazole, triazole, imidazole, or benzimidazole.

As used herein “heterocyclic or heterocyclyl” (on its own or in anycombination, such as “heterocyclylalkyl”)—a saturated or partiallyunsaturated 4-10 membered ring system in which one or more rings containone or more heteroatoms selected from the group consisting of N, O, orS; such as, but not limited to, pyrrolidine, piperidine, piperazine,morpholine, tetrahydro pyran, or imidazolidine.

As used herein, the expression “C₁₋₆ perfluoroalkyl” means that all ofthe hydrogen atoms in said alkyl group are replaced with fluorine atoms.Illustrative examples include trifluoromethyl and pentafluoroethyl, andstraight-chained or branched heptafluoropropyl, nonafluorobutyl,undecafluoropentyl and tridecafluorohexyl groups. Derived expression,“C₁₋₆perfluoroalkoxy”, is to be construed accordingly.

As used herein, the expression “C₃₋₈cycloalkyl” means cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.

As used herein, the expression “C₃₋₈cycloalkylC₁₋₆alkyl” means that theC₃₋₈cycloalkyl as defined herein is further attached to C₁₋₆alkyl asdefined herein. Representative examples include cyclopropylmethyl,1-cyclobutylethyl, 2-cyclopentylpropyl, cyclohexylmethyl,2-cycloheptylethyl and 2-cyclooctylbutyl and the like.

As used herein “halogen” or “halo” means chloro, fluoro, bromo, andiodo.

As used herein “C₁₋₆alkylsulfonyl” in the present context designates agroup—S(═O)₂C₁₋₆alkyl wherein C₁₋₆alkyl is as defined above.Representative examples include, but are not limited to, methylsulfonyl,ethylsulfonyl, n-propylsulfonyl, isopropylsulfonyl, butylsulfonyl,iso-butylsulfonyl, sec-butylsulfonyl, tert-butylsulfonyl,n-pentylsulfonyl, isopentylsulfonyl, neopentylsulfonyl,tert-pentylsulfonyl, n-hexylsulfonyl, isohexylsulfonyl and the like.

As used herein “arylsulfonyl” represents a group —S(═O)₂aryl whereinaryl is as defined above.

As used herein “heteroarylsulfonyl” represents a group —S(═O)₂heteroarylwherein heteroaryl is as defined above.

The expression “stereoisomers” is a general term used for all isomers ofthe individual molecules that differ only in the orientation of theiratoms in space. Typically it includes mirror image isomers that areusually formed due to at least one asymmetric center, (enantiomers).Where the compounds according to the invention possess two or moreasymmetric centers, they may additionally exist as diastereoisomers,also certain individual molecules may exist as geometric isomers(cis/trans). It is to be understood that all such isomers and mixturesthereof in any proportion are encompassed within the scope of thepresent invention.

“Substituted” means substituted by 1 to 2 substituents independentlyselected from the group consisting of C₁₋₆alkyl, C₁₋₆perfluoroalkyl,hydroxy, —CO₂H, an ester, an amide, C₁-C₆ alkoxy, C₁-C₆perfluoroalkoxy,—NH₂, Cl, Br, I, F, —NH-lower alkyl, and —N(lower alkyl)₂.

The compounds and salts of the present invention may exist in severaltautomeric forms, including the enol and imine form, and the keto andenamine form and geometric isomers and mixtures thereof. All suchtautomeric forms are included within the scope of the present invention.Tautomers exist as mixtures of a tautomeric set in solution. In solidform, usually one tautomer predominates. Even though one tautomer may bedescribed, the present invention includes all tautomers of the presentcompounds.

As used herein the term “modulator” refers to a chemical compound withcapacity to either enhance (e.g., “agonist” activity) or inhibit (e.g.,“antagonist” activity) a functional property of biological activity orprocess (e.g., enzyme activity or receptor binding); such enhancement orinhibition may be contingent on the occurrence of a specific event, suchas activation or repression of a signal transduction pathway and/or maybe manifest only in particular cell types and may result in a measurablebiological change.

As used herein, “patient” means a warm blooded animal, such as forexample rat, mice, dogs, cats, guinea pigs, and primates such as humans.

As used herein, the expression “pharmaceutically acceptable carrier”means a non-toxic solvent, dispersant, excipient, adjuvant, or othermaterial which is mixed with the compound of the present invention inorder to permit the formation of a pharmaceutical composition, i.e., adosage form capable of administration to the patient. One example ofsuch a carrier is a pharmaceutically acceptable oil typically used forparenteral administration.

The term “pharmaceutically acceptable salts” as used herein means thatthe salts of the compounds of the present invention can be used inmedicinal preparations. Other salts may, however, be useful in thepreparation of the compounds according to the invention or of theirpharmaceutically acceptable salts. Suitable pharmaceutically acceptablesalts of the compounds of this invention include acid addition saltswhich may, for example, be formed by mixing a solution of the compoundaccording to the invention with a solution of a pharmaceuticallyacceptable acid such as hydrochloric acid, hydrobromic acid, sulfuricacid, methanesulfonic acid, 2-hydroxyethanesulfonic acid,p-toluenesulfonic acid, fumaric acid, maleic acid, hydroxymaleic acid,malic acid, ascorbic acid, succinic acid, glutaric acid, acetic acid,salicylic acid, cinnamic acid, 2-phenoxybenzoic acid, hydroxybenzoicacid, phenylacetic acid, benzoic acid, oxalic acid, citric acid,tartaric acid, glycolic acid, lactic acid, pyruvic acid, malonic acid,carbonic acid or phosphoric acid. The acid metal salts such as sodiummonohydrogen orthophosphate and potassium hydrogen sulfate can also beformed. Also, the salts so formed may present either as mono- or di-acidsalts and can exist either as hydrated or can be substantiallyanhydrous. Furthermore, where the compounds of the invention carry anacidic moiety, suitable pharmaceutically acceptable salts thereof mayinclude alkali metal salts, e.g. sodium or potassium salts; alkalineearth metal salts, e.g. calcium or magnesium salts; and salts formedwith suitable organic ligands, e.g. quaternary ammonium salts.

The term “therapeutically effective amount” as used herein means anamount of the compound which is effective in treating the named disorderor condition.

The invention also provides pharmaceutical compositions comprising oneor more of the compounds according to this invention in association witha pharmaceutically acceptable carrier. Preferably these compositions arein unit dosage forms such as tablets, pills, capsules, powders,granules, sterile parenteral solutions or suspensions, metered aerosolor liquid sprays, drops, ampoules, auto-injector devices orsuppositories; for oral, parenteral, intranasal, sublingual or rectaladministration, or for administration by inhalation or insufflation.Alternatively, the compositions may be presented in a form suitable foronce-weekly or once-monthly administration; for example, an insolublesalt of the active compound, such as the decanoate salt, may be adaptedto provide a depot preparation for intramuscular injection. An erodiblepolymer containing the active ingredient may be envisaged. For preparingsolid compositions such as tablets, the principal active ingredient ismixed with a pharmaceutical carrier, e.g. conventional tabletingingredients such as corn starch, lactose, sucrose, sorbitol, talc,stearic acid, magnesium stearate, dicalcium phosphate or gums, and otherpharmaceutical diluents, e.g. water, to form a solid preformulationcomposition containing a homogeneous mixture of a compound of thepresent invention, or a pharmaceutically acceptable salt thereof. Whenreferring to these preformulation compositions as homogeneous, it ismeant that the active ingredient is dispersed evenly throughout thecomposition so that the composition may be readily subdivided intoequally effective unit dosage forms such as tablets, pills and capsules.This solid preformulation composition is then subdivided into unitdosage forms of the type described above containing from 0.1 to about500 mg of the active ingredient of the present invention. Flavored unitdosage forms contain from 1 to 100 mg, for example 1, 2, 5, 10, 25, 50or 100 mg, of the active ingredient. The tablets or pills of the novelcomposition can be coated or otherwise compounded to provide a dosageform affording the advantage of prolonged action. For example, thetablet or pill can comprise an inner dosage and an outer dosagecomponent, the latter being in the form of an envelope over the former.The two components can be separated by an enteric layer which serves toresist disintegration in the stomach and permits the inner component topass intact into the duodenum or to be delayed in release. A variety ofmaterials can be used for such enteric layers or coatings, suchmaterials including a number of polymeric acids and mixtures ofpolymeric acids with such materials as shellac, cetyl alcohol andcellulose acetate.

The liquid forms in which the novel compositions of the presentinvention may be incorporated for administration orally or by injectioninclude aqueous solutions, suitably flavored syrups, aqueous or oilsuspensions, and flavored emulsions with edible oils such as cottonseedoil, sesame oil, coconut oil or peanut oil, as well as elixirs andsimilar pharmaceutical vehicles. Suitable dispersing or suspendingagents for aqueous suspensions include synthetic and natural gums suchas tragacanth, acacia, alginate, dextran, sodium carboxymethylcellulose,methylcellulose, polyvinyl-pyrrolidone or gelatin.

In the treatment of various disease states as described herein, asuitable dosage level is about 0.01 to 250 mg/kg per day, preferablyabout 0.05 to 100 mg/kg per day, and especially about 0.05 to 20 mg/kgper day. The compounds may be administered on a regimen of 1 to 4 timesper day.

As used in the examples and preparations that follow, the terms usedtherein shall have the meanings indicated: “kg” refers to kilograms, “g”refers to grams, “mg” refers to milligrams, “g” refers to micrograms,“pg” refers to picograms, “mol” refers to moles, “mmol” refers tomillimoles, “nmole” refers to nanomoles, “L” refers to liters, “mL” or“ml” refers to milliliters, “μL” refers to microliters, “° C.” refers todegrees Celsius, “R_(f)” refers to retention factor, “mp” or “m.p.”refers to melting point, “dec” refers to decomposition, “bp” or “b.p.”refers to boiling point, “mm of Hg” refers to pressure in millimeters ofmercury, “cm” refers to centimeters, “nm” refers to nanometers, “[α]²⁰_(D)” refers to specific rotation of the D line of sodium at 20° C.obtained in a 1 decimeter cell, “c” refers to concentration in g/mL,“THF” refers to tetrahydrofuran, “DMF” refers to dimethylformamide,“NMP” refers to 1-methyl-2-pyrrolidinone, “brine” refers to a saturatedaqueous sodium chloride solution, “M” refers to molar, “mM” refers tomillimolar, “M” refers to micromolar, “nM” refers to nanomolar, “TLC”refers to thin layer chromatography, “HPLC” refers to high performanceliquid chromatography, “HRMS” refers to high resolution mass spectrum,“CIMS” refers to chemical ionization mass spectrometry, “ESI” refers toelectrospray ionization mass spectrometry, “t_(R)” refers to retentiontime, “lb” refers to pounds, “gal” refers to gallons, “L.O.D.” refers toloss on drying, “μCi” refers to microcuries, “i.p.” refers tointraperitoneally, “i.v.” refers to intravenously.

In one aspect of this invention there is disclosed novel compoundshaving the general structure shown in formula I:

whereinARYL is phenyl or pyridinyl, wherein said phenyl or pyridinyl isoptionally substituted with one or more substituents selected from thegroup consisting of halogen, C₁₋₆alkyl, C₂₋₆alkenyl, C₁₋₆alkoxy,C₁₋₆perfluoroalkyl; C₁₋₆alkylthio, hydroxy, hydroxyC₁₋₆alkyl,C₁₋₄acyloxy, nitro, cyano, C₁₋₆alkylsulfonyl, amino, C₁₋₆alkylamino andC₁₋₆alkoxycarbonyl;Z is —O(CH₂)_(n)—, —SO₂(CH₂)_(n)—, —(CH₂)_(n)—Y—(CH₂)_(n)—,—(CH₂)_(n)—CO—, —O(CH₂)_(n)—CO—, or —(CH₂)_(n)—Y—(CH₂)_(n)—CO— wherein Yis NR₃, O or S and R₃ is selected from the group consisting of H,C₁₋₆alkyl C₃₋₈cycloalkyl, C₁₋₆alkylC₃₋₈cycloalkyl and benzyl and n isindependently an integer from 1 to 5;X is NR₃, O or S wherein R₃ is as defined above;R₁ is H, halogen, C₁₋₆alkyl, C₁₋₆alkoxy, C₁₋₆perfluoroalkyl;hydroxyC₁₋₆alkyl, nitro, cyano, and C₁₋₆alkylamino; andR₂ is substituted or unsubstituted phenyl, pyridinyl or thienyl whereinthe substituents are selected from the group consisting of halogen,C₁₋₆alkyl, C₂₋₆alkenyl, C₁₋₆alkoxy, C₁₋₆perfluoroalkyl, C₁₋₆alkylthio,hydroxy, hydroxyC₁₋₆alkyl, C₁₋₄acyloxy, nitro, cyano, C₁₋₆alkylsulfonyl,amino, C₁₋₆alkylamino and C₁₋₆alkoxycarbonyl;with the proviso that when Z is —O(CH₂)_(n)— or —SO₂(CH₂)_(n)—, and ARYLis phenyl then R₂ is other than phenyl;or a stereoisomer, a tautomer or a solvate thereof or a pharmaceuticallyacceptable salt thereof.

In a further aspect of this embodiment, is disclosed a compound whereinARYL is phenyl and X is O or S.

In another aspect of this embodiment, is disclosed a compound wherein Xis O.

A compound exemplary of this embodiment is5-(4-{2-[5-methyl-2-(4-trifluoromethyl-phenyl)-thiazol-4-yl]-ethoxy}-phenyl)-3H-[1,3,4]oxadiazol-2-one.

In another embodiment of the present invention, is disclosed apharmaceutical composition comprising an effective amount of a compoundof formula I and a pharmaceutical acceptable carrier.

In another embodiment of the present invention, is disclosed a method oftreating a disease in a mammal wherein the disease is capable of being,modulated by PPARdelta ligand binding activity, which comprisesadministering to said mammal having said disease a therapeuticallyeffective amount of a compound of formula I.

whereinARYL is phenyl or pyridinyl, wherein said phenyl or pyridinyl isoptionally substituted with one or more substituents selected from thegroup consisting of halogen, C₁₋₆alkyl, C₂₋₆alkenyl, C₁₋₆alkoxy,C₁₋₆perfluoroalkyl; C₁₋₆alkylthio, hydroxy, hydroxyC₁₋₆alkyl,C₁₋₄acyloxy, nitro, cyano, C₁₋₆alkylsulfonyl, amino, C₁₋₆alkylamino andC₁₋₆alkoxycarbonyl;Z is —O(CH₂)_(n)—, —SO₂(CH₂)_(n)—, —(CH₂)_(n)—Y—(CH₂)_(n)—(CH₂)_(n)—CO—,—O(CH₂)_(n)—CO— or —(CH₂)_(n)—Y—(CH₂)_(n)—CO— wherein Y is NR₃, O or Sand R₃ is selected from the group consisting of H, C₁₋₆alkylC₃₋₈cycloalkyl, C₁₋₆alkylC₃₋₈cycloalkyl and benzyl and n isindependently an integer from 1 to 5;X is NR₃, O or S wherein R₃ is as defined above;R₁ is H, halogen, C₁₋₆alkyl, C₁₋₆alkoxy, C₁₋₆perfluoroalkyl;hydroxyC₁₋₆alkyl, nitro, cyano, and C₁₋₆alkylamino; andR₂ is substituted or unsubstituted phenyl, pyridinyl or thienyl whereinthe substituents are selected from the group consisting of halogen,C₁₋₆alkyl, C₂₋₆alkenyl, C₁₋₆alkoxy, C₁₋₆perfluoroalkyl, C₁₋₆alkylthio,hydroxy, hydroxyC₁₋₆alkyl, C₁₋₄acyloxy, nitro, cyano, C₁₋₆alkylsulfonyl,amino, C₁₋₆alkylamino and C₁₋₆alkoxycarbonyl; or a stereoisomer, atautomer or a solvate thereof or a pharmaceutically acceptable saltthereof.

In a further aspect of this embodiment, of the method of the inventionis disclosed a compound wherein ARYL is phenyl.

In another aspect of this embodiment, of this method of the invention isdisclosed a compound wherein ARYL is phenyl and R₂ is phenyl.

In a further aspect of this embodiment, of this method of the inventionis disclosed a compound wherein ARYL is phenyl, Z is —O(CH₂)_(n)— and R₂is phenyl.

In yet another aspect of this embodiment, of this method of theinvention is disclosed a compound wherein ARYL is phenyl, Z is—O(CH₂)_(n)—, X is O or S and R₂ is phenyl.

In another aspect of this embodiment, of this method of the invention acompound wherein ARYL is phenyl, Z is —O(CH₂)_(n)—, X is O or S, R₁ isC₁ alkyl and R₂ is phenyl.

In a further aspect of this embodiment, of this method of the inventionis disclosed a compound wherein X is O.

In yet another aspect of this invention, of this method of the inventionis disclosed a compound wherein X is S.

In a further aspect of this embodiment, is disclosed a method whereinsaid disease is a demyelinating disease selected form the groupconsisting of multiple sclerosis, Charcot-Marie-Tooth disease,Pelizaeus-Merzbacher disease, encephalomyelitis, neuromyelitis optica,adrenoleukodystrophy, Guillian-Barre syndrome and disorders in whichmyelin forming glial cells are damaged including spinal cord injuries,neuropathies and nerve injury.

In another aspect of this embodiment, is disclosed a method wherein thedemyelinating disease is multiple sclerosis.

In still another aspect of this invention is disclosed a method whereinsaid disease is selected from the group consisting of obesity,hypertriglyceridemia, hyperlipidemia, hypoalphalipoproteinemia,hypercholesterolemia, dyslipidemia, Syndrome X, Type II diabetesmellitus and complications thereof selected from the group consisting ofneuropathy, nephropathy, retinopathy and cataracts, hyperinsulinemia,impaired glucose tolerance, insulin resistance, atherosclerosis,hypertension, coronary heart disease, peripheral vascular disease orcongestive heart failure.

The compounds disclosed herein can be synthesized according to thefollowing procedures of Schemes, wherein the Aryl, X, Z and Rsubstituents are as identified for formula (I), above unless otherwisenoted. If necessary, in the following synthetic schemes, reactivefunctional groups present in the compounds described in this inventionmay be protected by suitable protecting groups. The protecting group maybe removed at a later stage of the synthesis. Procedures for protectingreactive functional groups and their subsequent removal may be found inT. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis,Wiley and Sons, 1991.

Scheme A shows the synthesis of the appropriate imidazole, oxazole orthiazole, intermediates for compounds of formula I wherein X is O, S orNR₃. The heterocycles can be prepared using methods known in thechemical literature (for reviews see Katritzky, A. R.; Rees, C. W. Eds.Comprehensive Heterocyclic Chemstry, Vol. 5; Pergamon Press (1984);Katritzky, A. R.; Rees, C. W.; Scriven, E. F. V. Eds. ComprehensiveHeterocyclic Chemstry II; Vols 3 & 4, Pergamon Press (1996)).Specifically, said oxazoles, imidazoles and thiazoles can be prepared byfusion of an appropriate α halo-ketone 1, respectively, with an amide,amidine or a thioamide (general formula 2), at temperatures ranging fromabout 40° C. to 150° C. to give the intermediate heterocycles 3.

In Scheme B the general synthesis of compounds of formula I wherein Z is—O(CH₂)_(n)— is shown. Accordingly, in Step B1 the appropriatelysubstituted carboxylic acid ester 4, which can be synthesized asillustrated in Scheme A is reduced to the alcohol 5 by methods that arewell known in the art. For example, the reduction may be effected byaluminum hydrides such as lithium aluminum hydrides ordiisobutylaluminum hydride in an inert solvent. In Step B2, the alcoholfunctional group in compound 5, is converted to a leaving group to givecompound 6, wherein Lg is a leaving group such as halogen, or sulfonateesters, for example mesylates or tosylates. Conversion to the leavinggroup can be accomplished by reaction of the alcohol with reagents suchN-bromosuccinimide in the presence of triphenylphosphine to produce acompound wherein the leaving group is bromide, or reaction with thionylchloride to give a compound wherein the leaving group is chloride. If asulfonate ester is desired, reaction of compound 5 with an appropriatesulfonyl chloride in the presence of a suitable base would produce thedesired sulfonate ester. For example, reaction of compound 5 withmethanesulfonyl chloride in the presence of an organic base such astriethylamine or pyridine in an inert solvent would give compound 6wherein the leaving group is OSO₂CH₃.

In Step B3 an appropriately substituted hydroxy aryl ester, 7 is reactedwith the heterocycle, 6 to displace the leaving group to afford coupledester, 8. The displacement reaction is run under conditions well knownin the art. Typically the reaction is run in the presence of a base suchas sodium hydride or other inorganic bases such as alkali carbonates oralkali hydroxides in an inert solvent. The temperature of the reaction,although not critical, is from 0° C. to the reflux temperature of theinert solvent.

Compound 8, in Step B4 is then treated with hydrazine either neat or ina suitable organic solvent at elevated temperatures to give the acidhydrazide, 9. Typically the reaction is run at a temperature of between50° C. and the reflux temperature of the organic solvent.

Cyclization of the acid hydrazide 9, in Step B5, to the target1,3,4-oxadiazol-2-ones, 10 is accomplished by treatment of compound 9with a chloroformate in the presence of an organic base such as pyridinefollowed by treatment with a strong, hindered amine base such as1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in a suitable organic solventsuch as acetonitrile in a sealed tube at elevated temperature.Typically, the reaction can be run from 100° C. to 200° C. The1,3,4-oxadiazol-2-ones may also be synthesized by reacting compound 9with phosgene. See Stempel, A., et al., J. Org. Chem. 1955, 20, 412.

In Step B6, an alternative synthesis of the coupled ester, 8 isillustrated. Accordingly, the alcohol, 5 can be reacted with thehydroxyaryl ester, 8 in the presence of a triaryl or trialkylphosphine,such as triphenylphosphine or tri- n-butylphosphine anddiethylazodicarboxylate in an inert solvent, for example THF ordichloromethane to afford the coupled ester 8. Typically the reaction isrun at a temperature between room temperature and the reflux temperatureof the inert solvent.

Scheme C illustrates the synthesis of the compound of formula I whereinZ is —(CH₂)_(n)—Y—(CH₂)_(n)—. The scheme is most useful to synthesizecompounds wherein n represents 1 or 2 in the alkylene chain attached toARYL. In Step C1 compound 5 (Y═O) is converted to compound 6 (wherein Lgis chloro or bromo) as described in Scheme B, Step B2. Compound 6 isthen reacted with thiourea, compound 11, under conditions similar tothose found in Treau, M. et al. Heterocycles, 2001, 55 (9), 1727-1735,to produce the thiol, 5a.

When compound 6 is reacted with a primary amine 12, the aminoalkylheterocycle 5b is produced. This displacement of a leaving group by anamine is well known to those skilled in the art. Typically, thedisplacement reaction is run in a polar organic solvent in the presenceof an organic base that acts as an acid scavenger. Although not criticalthe displacement reaction is run at a temperature of between ambient toreflux temperature of the solvent.

In Step C3 compounds 5, 5a and 5b can be reacted with compound 13 toafford the coupled arylester, 14, wherein Y is O, S or NR₃. Thus, whencompounds 5 (Y═O) and 5a (Y═S) are reacted with 13 to displace theleaving group, the reaction will typically be run in the presence of astrong base, for example sodium hydride, in a polar aprotic solvent,such as DMF or DMSO at temperatures of about between 0° C. to 150° C.When compound 5b (Y═NR₃) is reacted with 14, conditions identical tothose described above in Step C2 for the primary amine are used.

Synthesis of the desired 1,3,4-oxadiazol-2-ones 16, from compound 14 isaccomplished in the two steps (C4 and C5) exactly as described in SchemeB, Steps B4 and B5.

In Scheme D an alternative approach to compounds of formula I wherein Zis —(CH₂)_(n)—Y—(CH₂)_(n)— is shown. The scheme is most useful tosynthesize compounds wherein n represents 3 to 5 in the alkylene chainattached to ARYL.

In Step D1 the terminal aldehyde compound 17, which can be synthesizedby the method described in Scheme A, is converted in a two-step reactionsequence to the terminal acetylene, 19. Thus, reaction of 17 withbromomethylenetriphenylphosphorane (first step) with potassium t-BuOKproduces an intermediate bromoolefin (not shown), which is subsequentlytreated with 2 equivalents of t-BuOK (second step) to the form theacetylene, 19. The reaction sequence for the conversion is described inPianetti, P., Tet. Letters, 1986, 48, 5853-5856. Also, see Corey, E. J.,et al. J. Am. Chem. Soc., 1969, 91, 4318-4320. Alternatively, as shownin Step D2 intermediates of the type 19 can be prepared by displacementof a leaving group from an intermediate such as 6 (see Scheme C) using anucleophile, such as 18, wherein a terminal acetylene is incorporated.

In Step D3, Sonogashira coupling of acetylenic intermediate, 19 with thearyl iodide, 20 is effected in the presence oftetrakistriphenylphosphinepalladium (0), cuprous iodide and a suitableorganic base in an inert solvent to yield the coupled terminal acetylene21. The reduction of the acetylene, 21 can then be accomplished in StepD4 by catalytic hydrogenation of compound 21 to give the saturated ester14. Typically, the reduction can be accomplished by use of catalystssuch as palladium on carbon or chlorotris(triphenylphosphine)rhodium(I)in an inert organic solvent with hydrogen at pressures between 30 to 300p.s.i. The reduction can be run at a temperature between roomtemperature and 175° C.

Synthesis of the desired 1,3,4-oxadiazol-2-ones 16, from compound 14 isaccomplished in two steps (D5 and D6) exactly as described in Scheme B,Steps B4 and B5.

Scheme E illustrates a particular synthesis of compounds of formula Iwherein Z is —(CH₂)_(n)NR₃(CH₂)_(n)—. In this approach, the linker Z isconstructed by a reductive amination of an aldehyde with an amine. Forexample, in Step E1 treatment of 5b (wherein Y═NR₃) with an aldehyde,such as 4-formyl-benzoic acid methyl ester (n═1) compound 22, in a polarsolvent, usually an alcohol or an alcohol THF mixture, followed bytreatment with a reducing agent such as sodium triacetoxy-borohydrideprovides the required intermediate 14a (n═1).

Similarly, in Step E2 treatment of an aldehyde such as 17a with anamine, such as 4-aminoalkyl benzoic acid methyl ester (n═1), compound23, provides 14a, wherein n is 1 and R₃ is H for —(CH₂)_(n)NR₃. Compound14a in steps E3 and E4 is converted to 1,3,4,-oxadiazol-2-ones 16a asdescribed in Scheme B, Steps B4 and B5.

More generally, appropriate amines (R′OOC-ARYL-(CH₂)_(n)NHR₃) areprepared from the corresponding nitriles or nitro compounds by catalytichydrogenation or from acetylenic amines and an aryl iodide or bromide bySonogashira coupling followed by catalytic hydrogenation as described inScheme D.

Scheme F illustrates the synthesis of compounds of formula I wherein Zis —SO₂ (CH₂)_(n)—. In Step F1 treatment of an aryl sulfonyl chloride,24 with aqueous sodium sulfite provides the sulfinic acid, 25. Reactionof 25, as in Step F2, with an intermediate such as 6 in a polar solventsuch as DMF, acetonitrile or ethanol in the presence of a base such asDBU, pyridine, sodium methoxide or sodium hydroxide providesintermediate 26. Intermediate 26 is converted to the corresponding1,3,4-oxadiazol-2-one, 28 in Steps F3 and F4 as illustrated in Scheme B,Steps B4 and B5.

Scheme G illustrates the synthesis of compound of formula I wherein Z is—O(CH₂)_(n)CO—. The scheme illustrates the case wherein n is 1. Thestarting 2-acyl heterocycle, 29 can be synthesized from thecorresponding carboxylic acid (prepared by the method illustrated inScheme A) by addition of an appropriate Grignard reagent to anintermediate N-methoxy N-methyl carboxamide (Khlestkin, V. K. et al.;Current Organic Chemistry, 2003, 7(10), 967-993. and Singh, J. et al.,Journal für Praktische Chemie, 2000, 342, 340-347). Preparation of theintermediate N-methoxy-N-methyl carboxamide is most conveniently carriedout by reaction of the acid with N-methoxy-N-methyl hydroxylaminehydrochloride in the presence of a peptide coupling reagent such as EDC,DCC, DMPU and a tertiary amine base such as diisopropylethylamine ortriethylamine.

Thus in hand, 29 is brominated to produce the bromoketone 30, as shownin Step G1. The bromination can be accomplished by well-known methods,for example reaction of 29 with pyridinium bromide per bromide in aceticacid or reaction of 29 with Br₂ in an inert organic solvent such asdichloromethane. The resulting bromoketone 30, in Step G2, is reactedwith the arylhydroxy ester 7 under conditions described in Scheme B(Step B3) to afford the coupled ester 31. The ketone functionality in 31is protected as a ketal 32, as shown in Step G3 by methods well known inthe art. Compound 32 is then converted to the 1,3,4-oxadizol-2-one ketal34 in Steps G4 to G5 by the standard sequence as described in Scheme B(B4 and B5). Finally, in Step G6, the ketal functionality in 34 iscleaved, for example, with mineral acid in THF-methanol-water or othermethods known in the art to afford the target structure 35.

It would be evident to one skilled in the art that the above procedureof Scheme G could be used to synthesize analogs where n is 2-5 forcompound 35 by starting with a bromoketone, compound 30, with a largerbromoalkanoyl substituent (Br(CH₂)_(n)CO—, wherein n is 2 to 5).

Scheme H illustrates the procedure for the preparation of compounds offormula I wherein Z is —(CH₂)_(n)CO—. In Step H1, the appropriatemethoxycarbonyl-substituted heterocycle, 36 is treated with 2equivalents of the lithium enolate of t-butylacetate in a solvent suchas THF or DME at a temperature ranging from −78° C. to room temperatureto provide the ketoacetate intermediate 37. In Step H2, treatment of 37with a base such as sodium hydride in an inert solvent at a temperaturebetween −10° C. and room temperature followed by alkylation of theresulting anion with an electrophile such as 13 yields the advancedintermediate ketodiester 38. The decarboxylation shown in Step H3 andcan be accomplished by first treatment of 38 with TFA in an inertsolvent such as dichloromethane followed by thermolysis at a temperaturebetween 70° C. and 150° C. to provide intermediate ketoester 39. Theketone functionality in 39 is protected as a ketal 40, as shown in StepH4 by methods well known in the art. Compound 40 is then converted tothe 1,3,4-oxadiazol-2-one ketal 42 in Steps H5 to H6 by the standardsequence as described in Scheme B (B4 and B5). Finally, in Step H7, theketal functionality in 42 is cleaved, as described above in Scheme G,Step G6 to afford the desired 1,3,4-oxadiazol-2-one, compound 43.

BIOLOGICAL EXAMPLES

The following test protocols are used to ascertain the biologicalproperties of the compounds of this invention. The following examplesare being presented to further illustrate the invention. However, theyshould not be construed as limiting the invention in any manner.

Determination of EC₅₀ Values in the Cell Based PPARdelta-GAL4 AssayPrinciple

The potency of substances, which bind to human PPAR delta and activateit in an agonistic manner, is analyzed using a stably transfected HEKcell line (HEK=human embryo kidney) which is referred to here as PPARdelta reporter cell line. The PPAR delta reporter cell line contains twogenetic elements, a luciferase reporter element (pdeltaM-GAL4-Luc-Zeo)and a PPAR delta fusion protein (GR-GAL4-humanPPAR delta-LBD), whichmediates expression of the luciferase reporter element depending on aPPAR delta ligand. The stably and constitutively expressed fusionprotein GR-GAL4-humanPPAR delta-LBD binds in the cell nucleus of thePPAR delta reporter cell line via the GAL4 protein portion to the GAL4DNA binding motifs 5′-upstream of the luciferase reporter element whichis stably integrated in the genome of the cell line. There is onlylittle expression of the luciferase reporter gene in the absence of aPPAR delta ligand if fatty acid-depleted fetal calf serum (cs-FCS) isused in the assay. PPAR delta ligands bind and activate the PPAR deltafusion protein and thereby stimulate expression of the luciferasereporter gene. The luciferase, which is formed can be detected by meansof chemiluminescence via an appropriate substrate.

Construction of the PPAR Delta Reporter Cell Line:

The production of the stable PPAR delta reporter cell line is based on astable HEK-cell clone which was stably transfected with a luciferasereporter element. This step was already described above in the section“construction of the PPAR alpha reporter cell line”. In a second step,the PPAR delta fusion protein (GR-GAL4-humanPPAR delta-LBD was stablyintroduced into this cell clone. For this purpose, the cDNA coding forthe N-terminal 76 amino acids of the glucocorticoid receptor (Accession#P04150) was linked to the cDNA section coding for amino acids 1-147 ofthe yeast transcription factor GAL4 (Accession #P04386). The cDNA of theligand-binding domain of the human PPAR delta receptor (amino acidsS139-Y441; Accession #L07592) was cloned in at the 3′-end of thisGR-GAL4 construct. The fusion construct prepared in this way(GR-GAL4-humanPPAR delta-LBD) was recloned into the plasmid pcDNA3(Invitrogen) in order to enable constitutive expression by thecytomegalovirus promoter. This plasmid was linearized with a restrictionendonuclease and stably transfected into the previously described cellclone containing the luciferase reporter element. The resulting PPARdelta reporter cell line which contains a luciferase reporter elementand constitutively expresses the PPAR delta fusion protein(GR-GAL4-human PPAR delta-LBD) was isolated by selection with zeocin(0.5 mg/ml) and G418 (0.5 mg/ml).

Assay Procedure and Evaluation:

The activity of PPAR delta agonists is determined in a 3-day assay,which is described below:

Day 1

The PPAR delta reporter cell line is cultivated to 80% confluence inDMEM (#41965-039, Invitrogen) which is mixed with the followingadditions: 10% cs-FCS (fetal calf serum; #SH-30068.03, Hyclone), 0.5mg/ml zeocin (#R250-01, Invitrogen), 0.5 mg/ml G418 (#10131-027,Invitrogen), 1% penicillin-streptomycin solution (#15140-122,Invitrogen) and 2 mM L-glutamine (#25030-024, Invitrogen). Thecultivation takes place in standard cell culture bottles (#353112,Becton Dickinson) in a cell culture incubator at 37° C. in the presenceof 5% CO₂. The 80%-confluent cells are washed once with 15 ml of PBS(#14190-094, Invitrogen), treated with 3 ml of trypsin solution(#25300-054, Invitrogen) at 37° C. for 2 min, taken up in 5 ml of theDMEM described and counted in a cell counter. After dilution to 500.000cells/ml, 35,000 cells are seeded in each well in a volume of 180 μL ofa 96 well microtiter plate with a clear plastic base (#3610, CorningCostar). The plates are incubated in the cell culture incubator at 37°C. and 5% CO₂ for 24 h.

Day 2

PPAR delta agonists to be tested are dissolved in DMSO in aconcentration of 10 mM. This stock solution is diluted in DMEM(#41965-039, Invitrogen) which is mixed with 5% cs-FCS (#SH-30068.03,Hyclone), 2 mM L-glutamine (#25030-024, Invitrogen) and the previouslydescribed antibiotics (zeocin, G418, penicillin and streptomycin). Testsubstances are tested in 11 different concentrations in the range from10 μM to 100 pM. More potent compounds are tested in concentrationranges from 1 μM to 10 pM or between 100 nM and 1 pM.

The medium of the PPAR delta reporter cell line seeded on day 1 iscompletely is either completely removed by aspiration or not, and thetest substances diluted in medium are immediately added to the cells.The dilution and addition of the substances is carried out by a robot(Beckman FX). The final volume of the test substances diluted in mediumis 100 μl per well of a 96 well microtiter plate. The DMSO concentrationin the assay is less than 0.1% v/v in order to avoid cytotoxic effectsof the solvent.

Each plate was charged with a standard PPAR delta agonist, which waslikewise diluted in 11 different concentrations, in order to demonstratethe functioning of the assay in each individual plate. The assay platesare incubated in an incubator at 37° C. and 5% CO₂ for 24 h.Alternatively, 20 μL of a 10× final concentration of the test substanceis added directly to the 180 μL containing the plated cells. The testsubstances are tested in 8 different concentrations, in triplicate, inthis assay plate set-up.

Day 3

The PPAR delta reporter cells treated with the test substances areremoved from the incubator, and the medium is aspirated off. The cellsare lyzed by pipetting 50 μl of Bright Glo reagent (from Promega) intoeach well of a 96 well microtiter plate. After incubation at roomtemperature in the dark for 10 minutes, the microtiter plates aremeasured in the luminometer (Trilux from Wallac). The measuring time foreach well of a microtiter plate is 1 sec.

Evaluation:

The raw data from the luminometer are transferred into a Microsoft Excelfile. Dose-effect plots and EC₅₀ values of PPAR agonists are calculatedusing the XL.Fit program as specified by the manufacturer (IDBS).

PPARdelta EC₅₀ values in the range of 1 nM to >10 μM were measured forthe PPAR modulators of the examples in this application. Compounds ofthe invention of formula I can act as agonists or antagonists. The assayto determine partial agonist or antagonist activity is described below.

Determination of Effectiveness of Partial Agonists or Antagonists At thePPARdelta Receptor

This assay determines if compounds act as partial agonists orantagonists at the PPARdelta receptor.

The plating and harvesting of the assay plates is as described in Day 1and 3 above.

Day 2

The partial agonist or antagonist and a known selective agonist arediluted in DMEM (#41965-039, Invitrogen), which is mixed with 10% cs-FCS(#SH-30068.03, Hyclone), 2 mM L-glutamine (#25030-024, Invitrogen) andthe previously described antibiotics (zeocin, G418, penicillin andstreptomycin) to 20× desired final concentrations. Ten microliters ofthe partial agonist or antagonist is added to the cell-containing assayplate. The assay plates are incubated in an incubator at 37° C. and 5%CO₂ for 30 minutes. Ten microliters of the 20× known selective agonistsare then added, after the partial agonist or antagonist pre-incubation.The assay plates are incubated in an incubator at 37° C. and 5% CO₂ for24 h. The effect on the known selective agonists EC₅₀'s is determinedfor each partial agonist or antagonist concentration.

SPA PPARdelta-LBD Binding Assay Stock Solutions:

1 M Tris (pH=8.0 or pH=7.6)(Gene Medicine Stock Room)

2 M KCl (Powder in N2140)

Tween 20

100 mM DTT

13.9 uM GW2331 in EtOH HOT

10 mM GW2331 in DMSO COLD

PPAR-alpha (Conc. varies)

Ex: 0.884 μg/μl

Wash Buffer: ( Store at 4° C. Buffer is good for one week)

10 mM Tris(pH = 7.6 or 8) 10 ml 50 mM Kcl 25 ml 0.05% Tween 20 0.5 ml Millipore Water 964.5 Check PH = 7.6Binding Buffer: ( Prepare fresh binding buffer every time)

Wash Buffer  50 ml 10 mM DTT 5.5 ml

Preparation of Reaction Reagents for 1 Plate:

Glutathione Coated SPA beads

Each SPA bead bottle contains 500 mg beads

Reconstitute 500 mg of SPA beads in 5 ml of wash buffer, and will begood for few weeks) Store at 4° C.

Prepare Diluted SPA Beads in the Binding Buffer

Adding 1 ml of above reconstituted SPA beads to 60 ml of Binding bufferadding 20 μl of above diluted beads to each well of a 96-well plate.

Use 2 ml of above diluted beads for each plate (no dead volumeincluded).

3H GW-2331 plus GST-PPAR delta-LBD (for one 96-well plate no deadvolume) 13.9 μM 40 nM/well

3.0 ml/plate (Including dead volume)

If 3H-GW2331 specific activity is 1 mci/ml(From Amersham), dilute 17 μlof 3H GW-2331 into 3.0 ml of Binding Buffer=0.08 μM

If protein concentration is 1 mg/mL, add 21 μμl of proteins into 3.0 mlof binding buffer.

In Summary: ONE 96-well plate: 3000 μL Binding Buffer+17 μL of3H-GW2331+21 μL of GST-PPAR-delta(1 mg/ml)

Control Plates

A 96-well Mother Plate (For 2 control Plates)

In column #1:

Add 5 μl of cold GW2331 (10 mM) to the wells E-H.

Add 45 μL of DMSO to the wells A-H.

In column #12 (3-fold dilution):

Add 10 μL of cold GW2331 (10 mM) to the well A.

then add 90 μl of DMSO to the well A, mix well the solution.

add 20 μl of DMSO to the wells B-H.

take 10 μl solution from the well A to B, mix well,

then take 10 μl solution from B to C, mix well,

then take 10 μl solution from C to D, mix well.

Finally, take 10 μl from F to H.

A Control Plate (for 8 Reaction Plates)

A control plate is 1:10 dilution of the mother plate. The dilutionbuffer is the wash buffer.

Sample Plates

To fresh CPC library plate, add 90 μl of DMSO

Take 10 μl of DMSO dilution and add it to 90 μl of wash buffer in asample plate

Reaction Plates:

Add 20 μl of SPA beads and 30 μl of 3H-GW2331 with GST-PPAR-delta toeach well of a

Reaction Plate.

Add 5 μl compounds from each well of the sample plate into columns 2 to11 of a reaction plate.

Add 5 μl compounds from column 1 and column 12 of the control plate tothe column 1 and column 12 of the reaction plate.

96-well SPA Protocol:

Let reaction plates equilibrate for 20 minutes to 2 hours.

Seal the plates before counting in a Microbeta counter (Wallac).

Calculate IC₅₀.

In the SPA PPAR delta-LBD Binding Assay IC₅₀ values in the range of 1 nMto >10 μM were measured for the PPAR modulators of the examples in thisapplication. Compounds of the invention of formula I can act as agonistsor antagonists.

RAT/MICE Oligodendrocyte Cultures Preparation of Cells:

1. Primary rat oligodendrocyte progenitor cells are obtained from theneocortex of newborn (postnatal days 2-3) rats or mice and are enriched,after removal of microglia, by mechanical separation from the astrocyticmonolayer using a modification of the technique originally described byMcCarthy and de Vellis (1980).

2. Remove the meninges from neonatal rat brain and mechanicallydissociate tissue. Plate cells on T75 flasks and feed cells withDMEM/F12+10% FBS.

3. Collect oligodendrocytes growing on the astrocyte bed layer byshaking-off method fourteen days after the original prep date.Centrifuge the suspension and resuspend the cell pellet in serum freemedia (SFM; DMEM combined with 25 μg/ml transferring, 30 nMtriiodothyronine, 20 nM hydrocortisone, 20 nM progesterone, 10 nMbiotin, 1× trace elements, 30 nM selenium, 1 μg/ml putrescine, 0.1% BSA,5 U/ml PenStrep, 10 μg/ml insulin) supplemented with the followinggrowth factors: Platelet derived growth factor-AA (PDGF) and fibroblastgrowth factor-2 (FGF).

4. Plate the cells on PDL-coated dishes and incubate at 37° C. with 6-7%CO₂.

5. Media components are replaced every 48 hr to keep the cells in aprogenitor state.

Progenitor Cell Passaging to Increase Cell Numbers for Screening Assays:

1. When the culture are confluent, rinse the culture with PBS, addtrypsin and incubate for 2-3 min at 37° C.

2. Neutralize and centrifuge the cell suspension at 900 g for 5 min.

3. Resuspend the cell pellet in SFM+PDGF/FGF.

4. Feed the cells with fresh growth factors every 48 hrs to keep enrichfor rapidly dividing progenitor cells.

5. Cells are passaged no more than 4-5 times prior to experimentalassays.

6. All experiments involving oligodendrocyte progenitor cells were doneusing cells that were continuously maintained under these conditions.Greater than 95% of all cells were A2B5 immunopositive and expressed2′3′-cyclic nucleotide 3′-phosphodiesterase II mRNA.

7. To generate mature oligodendrocytes, 24 h after plating progenitorcells were switched to SFM supplemented with or without IGF-I and grownunder these conditions for 7 d prior to experimental assays.

8. Alternatively, the enriched rat Central Glia-4 (CG4) progenitor cellline may be used, which is maintained in base media (DMEM, with 2 mMglutamine, 1 mM sodium pyruvate, biotin (40 nM), insulin (1 μM) and N1)supplemented with 30% conditioned media from the B-104 neuroblastomacell line. To induce differentiation, CG4 cells are switched to basemedia with 1% fetal calf serum (removed after 2 days) and insulin (500nM). A2B5 and MBP immunoreactivity is used to confirm >95% enrichment inimmature and mature cultures, respectively.

Rat/Mouse Culture Compound Treatment:

1. Put 10,000 - 15,000 cells /well in 24-well PDL coated plates andculture the cells in presence of mitogen (10 ng/ml) overnight.

2. In the presence of mitogen:

-   -   a. Next day, remove the old medium and add compounds in fresh        medium (with mitogen)    -   b. Compound dose response evaluations are performed at 6        different concentrations (10 μM, 1 μM, 100 nM, 10 nM, 1 nM, and        0.1 nM);    -   c. Triplicates wells are run for each compound concentration.

3. In the absence of mitogen:

-   -   a. Next day, remove the old medium and add compounds in fresh        medium (without mitogen)    -   b. Compound dose response evaluations are performed at 6        concentrations (10 μM, 1 μM, 100 nM, 10 nM, 1 nM, and 0.1 nM);    -   c. Triplicates wells are run for each compound concentration.

4. Culture the treated cells for 7 d prior to using in experimentalassays.

HUMAN Oligodendrocyte Cultures Preparation of Cells:

1. Human neurospheres collected from EI9.5-E22 human embryo cortex) arecultured for 2 weeks in progenitor media: DMEM/F12 containing 100 μg/mltransferring, 30 nM triiodothyronine, 20 nM hydrocortisone, 20 nMprogesterone, 10 nM biotin, 1× trace elements, 30 nM selenium, 60 uMputrescine, 0.1% BSA, 5 U/ml PenStrep, 25 μg/ml insulin) supplementedwith PDGF and FGF.

2. Neurospheres are dissociated with 20 U/ml papain at 37° C. for 30-50min.

3. Cells are plated onto PDL coated dishes at density of 50,000-100,000cell/well in progenitor media containing PDGF/FGF and incubated at 37°C. with 5-6% CO₂.

4. Media and growth factors are replenished every 48 hr.

Human Culture Compound Treatment:

1. 24 to 48 hr after plating remove the old medium and add compounds infresh medium (with mitogen)

2. Compound dose response evaluations are performed at 3-6 differentconcentrations (10 μM, 1 μM, 100 nM, 10 nM, 1 nM, and 0.1 nM)

3. Triplicates wells are run for each compound concentration.

5. Culture the treated cells for 7 d prior to using in experimentalassays.

RAT/MOUSE/HUMAN Oligodendrocyte Specific Immunostaining:

Following compound exposure, oligodendrocyte-specific antibodies areused to assess ability of compound to accelerate/promote oligodendrocytedifferentiation (for example, O4, O1, or myelin basic proteinimmunoreactivity is over time between compound treated and untreatedcultures).

1. Cells are plated onto poly-D-lysine treated 4-well chamber slides at5×10³ to 20×10³ cells/well and grown as described above. Sequentialstaining is performed on oligodendrocyte populations with increasingdegrees of cellular differentiation, as determined by days in vitrowithout PDGF and FGF.

2. Live staining for 30 min at 37° C. is used to detect oligodendrocytestage specific cell surface marker expression (including A2B5, O4, andO1).

3. Subsequently, cells are fixed with 4% paraformaldehyde, 10 min, roomtemperature.

4. Fixed staining procedures are used to detect oligodendrocyte stagespecific marker expression (including myelin basic protein, MBP).

5. Rinse with PBS.

6. Permeabilize with 0.1% Triton/0.01% NaAz diluted in 1× PBS for 10min, room temperature.

7. Block with 5-10% goat serum in antibody dilution buffer (0.1%Triton-X 100 and 1% IgG-free bovine serum albumin; also used to diluteantibodies), 15 min, room temperature.

8. Add primary antibody diluted in antibody dilution buffer.

9. Incubate overnight, gently rocking, 40° C.

10. Next day, rinse with PBS 1× 5 min, followed by 3× 15 min each, roomtemperature.

11. Incubate with appropriate secondary antibodies, 45 min, roomtemperature.

12. Cell nuclei are stained with 4,6-diamidino-2-phenylindole (DAPI), 15min, room temperature.

13. Rinse several times with PBS and evaluate using fluorescentmicroscopy.

14. The following conditions are compared over time and at differentcompound doses: PDGF/FGF alone, SFM alone, SFM-IGF 1 alone, PDGF/FGF andcompound, SFM and compound.

RAT/MOUSE/HUMAN Bromodeoxyuridine (BrdU) Immunostaining To confirm thatCompounds Do Not Promote Cell Proliferation

1. Oligodendrocyte progenitor cells are labeled with 10 μM BrdU for 20hr and then fixed with either 70% ethanol or 4% paraformaldehyde.

2. The cells are incubated successively with biotinylated mouseanti-BrdU and Streptavidin-Peroxidase, with three intervening washeswith PBS.

3. Colormetric visualization of the BrdU immunoreactivity is developedwith DAB and total cell numbers are assessed using the counter-stainhematoxylin.

4. BrdU immunopositive cells are counted by two independent observers.

RAT/MOUSE/HUMAN Culture Image analysis: Fluorescent microscopy is usedto quantitate the extent of oligodendrocyte differentiation aftercompound exposure. This assay demonstrates that selective agonistsaccelerate/promote oligodendrocytes differentiation.

1. Manual Cell Counting: Four fields are randomly selected for eachexperimental condition and 500-600 cells are counted in each field. Thepercentage of MBP (or O4) immunpositive cells (mature process bearingcells with or without myelin sheets) versus DAPI positive cells (totalcell number) cells are compared in the control and drug-treated groups.

2. Automated Cell Counting: Fluorescent microscopy was used toquantitate the extent of oligodendrocyte differentiation after compoundexposure. Six fields/well were randomly selected to assess the number ofdifferentiating oligodendrocytes among the total population (˜8 to15×10³ cells are counted/well). Immunofluorescence images were obtainedusing a Zeiss AxioVision digital imaging system, with a Zeiss AxioCamHRc cooled CCD camera connected to the same microscope. All microscopicimaging parameters were set for acquiring images for the analysis ofcellular immunofluorescence intensity. The percentage of MBP positive(differentiated) cells versus total cells (DAPI nuclear stained) wascompared in the control versus drug-treated groups. Cellularautofluorescence was undetectable under the imaging conditions.

3. Human oligodendrocyte differentiation assay: manually count totalnumber of O4 immunopositive cells/well (bipolar and multipolar).

RAT/MOUSE/HUMAN Quantitative Polymerase Chain Reaction (PCR): Toevaluate compound induced PPAR delta pathway activation and the extentof oligodendrocyte maturation (changes in mRNA levels).

1. Total RNA is extracted from cultured oligodendrocytes using TriZolreagent.

2. Subsequently, mRNA is treated with RNase-free DNase, repurified, andthen converted to cDNA template using a RT reaction (Clontech AdvantageRT for PCR Kit).

3. PPAR delta pathway member transcript expression is quantitated usingSybr Green PCR Master Mix.

4. The 18S ribosomal RNA primer/probe mix (186 bp product), suspended inTaqman 2× PCR Master Mix is used as an internal control.

5. Quantitative PCR is carried out using real-time Taqman™ technology(Gibson, et al., 1996) with a model 7700 Sequence Detector System(Applied Biosystems, Foster City, Calif.).

6. The results are analyzed using Sequence Detection Systems softwareversion 1.91.

RAT ELISA Assay: To evaluate compound induced PPAR delta pathwayactivation and the extent of oligodendrocyte maturation (changes inprotein levels).

1. Plates are washed with PBS, and then keep on ice. Add 200 μl ice oldlysis buffer (Tris 50 mM, pH7.4, MgCl2 2 mM, EDTA 1 mM,β-mercaptoethanol 5 mM, Nonidet P-40 1%, Protease inhibitor cocktail(Roche): 1 tablet/50 ml) to each well.

2. Lyse cells by using pipette to up down and spin plates at 2000 rpm at4° C. for 5 min.

The supernatant is ready to use.

3. Pipet 50 μl of standard, controls and samples to the wells.

4. Add 50 μl of MBP Assay Buffer to each well.

5. Incubate the well, shaking at 500-700 rpm on orbital microplateshaker for 2 hr at room temperature.

6. Add 100 μl of the MBP Antibody-Biotin Conjugate to each well.

7. Incubate the well, shaking at 500-700 rpm on orbital microplateshaker for 1 hr at room temperature.

8. Wash well 5 times with Wash Solution. Blot dry by inverting the plateon absorbent material.

9. Dilute the streptavidin-enzyme conjugate concentrate 1:50 with MBPElisa Assay buffer. (must be diluted immediately prior to use in theassay).

10. Add 100 μl streptavidin-enzyme conjugate solutions to each well.

11. Incubate the well, shaking at 500-700 rpm on orbital microplateshaker for 30 min at room temperature.

12. Wash well 5 times with the Wash Solution. Blot dry by inverting theplate on absorbent material.

13. Add 100 μl of TMB Chromogen Solution to each well.

14. Incubate the well, shaking at 500-700 rpm on orbital microplateshaker for 10-20 min at room temperature. Avoid exposure to directsunlight.

15. Add 100 μl of the Stopping Solution to each well.

Read the absorbance of the solution in the wells within 30 min, using amicroplate reader set to 450 nM

In Vivo Proof of Concept Models Focal Lesions: (Used to Assess WhetherCompounds Protect Myelin Integrity or Accelerate/enhance the Rate ofRemyelination.)

1. Rats 7 weeks of age are given free access to food and water andacclimatized for a minimum of 4 days before use in experiments.

2. Prior to surgery each animal is weighed. The rat is thenanaesthetized with ketamine (100 mg/ml) in combination with xylazine (20mg/ml) in a ratio of 1.8:1. The rats are injected with 0.15 ml/180 gbody weight i.p. of the anaesthetic solution prior to the surgicalprocedure. The animal is prepared for surgery using aseptic conditionsin accordance with the IACUC guidelines. All surgical instruments willbe autoclaved. The hair is clipped between the ears and this region willthen be scrubbed with Betadine, flushed with sterile saline and finallywiped with a pre-packaged sterile alcohol swab.

3. For the surgical procedure, the rat is placed on its ventral surfacein a small animal stereotaxic instrument designed to hold the headsteady. The incisor bar is always set at −3.9 mm, since this has beenshown to achieve a flat-skull position for SD rats.

4. An incision is made in the previously shaven skin overlying the skullbetween the ears.

5. A small area of bone (0.75 mm in diameter) is drilled at thefollowing coordinates AP—1.8, ML-3.1 from lambda.

6. The bone is removed and rats are injected with 2 μl ethidium bromide,lysolecithin, or SIN-1 into the right caudal cerebellar peduncle, DV-7.1 mm, over a 2 min period by means of a Hamilton μl syringe andneedle. Alternatively injections are made into the spinal cord, corpuscallosum, or cortex.

7. The needle is left in position for the subsequent 2 min.

8. After withdrawal of the needle the incision is sutured.

9. Each rat receives an i.m. injection of 0.003 mg buprenorphine into ahind leg.

10. The rat is placed in a warming cupboard until it regainsconsciousness. At which time it is returned to its home cage. Do notallow more than 2 rats per cage, as they will pull each other's sutureout.

11. Similar procedures are also done using mice.

Rat Experimental Allergic Encephalomyelitis (Rat EAE) Disease Model

Experimental allergic encephalomyelitis (EAE) is a T-cell-mediatedautoimmune disease of the nervous system that develops in susceptibleanimals following sensitization with either whole spinal cord homogenateor a component (myelin basic protein). The EAE rodent model is anappropriate tool for studying the inflammation of the brain and spinalcord observed in MS patients. In rodents, injection of whole spinal cordor spinal cord components such as myelin basic protein induces anautoimmune response based on the activation of T-lymphocytes. Clinicaldisease typically becomes manifest around day 8-10 after inoculation,observed as a broad spectrum of behavioral anomalies ranging from mildgait disturbances and tail atony to complete paralysis and death. Weightloss typically occurs. In animals that survive, spontaneous recoveryoccurs, accompanied by variable recovery of most motor function.Depending on the species, allergen, and methodology used, animals testedby the EAE model may experience a single (acute EAE) or several (chronicrelapsing EAE) attacks. Several treatment paradigms may be used: thedrug or treatment of choice may be administered before immunization,during the nonsymptomatic period or during the clinical disease.

Animals:

Female Lewis rats, 160-220 g (Charles River)

Antigen:

Whole Guinea Pig spinal cord (Harlan Biosciences).

Complete Freund's adjuvant H37 Ra [1 mg/ml Mycobacterium TuberculosisH37 Ra] (Difco).

Additional Antigen:

Mycobacterium Tuberculosis (Difco).

Bordetella Pertussis [Heat Killed] (Difco).

Antigen preparation: (for approximately 720 animals):

1. Weigh 5 grams of frozen guinea pig spinal cord.

2. Add 5 g spinal cord to 5 ml 0.9% saline (1 g/ml) in a round bottomcentrifuge tube

3. Homogenize on ice with the Tissue-tech until the tissue is completelydisrupted (approximately 5 minutes).

4. Add 10 ml Complete Freund's adjuvant H37 Ra supplemented with 200 mgMycobacterium Tuberculosis (20 mg/ml Complete Freund's adjuvant H37 Ra).

5. Extract homogenate/adjuvant from tube by sucking it into a 10 mlsyringe fitted with an 18 gauge emulsifying needle.

6. Emulsify between two 30 ml glass syringes until it becomes difficultto continue passing the material through the needle. (Approximately 5minutes {there must be no separation between the oil phase and theaqueous phase}).

7. Use immediately or keep on ice until needed (not more than 30 min)(do not freeze).

Protocol

1. Female Lewis rats (Charles River) are given free access to food andwater and should be acclimated a minimum of 3 days before use inexperiments.

2. Rats weighing 160 and 220 grams are initially induced with 5%isoflurane (Aerrane, Fort Dodge), 30% O₂, 70% N₂O for 2-5 minutes.

3. The rat is then placed onto a circulating water heating blanket(Gaymar) (dorsal surface up) and into the nose cone for spontaneousrespiration of anesthetic gases. The isoflurane is reduced to 2%.

4. Two subcutaneous injections (0.1 ml each) of either antigen or normalsaline are made into ventral surface of the hind paws.

5. The animals are removed from the nose cone, weighed and numbered.

6. The rats are allowed to awake from anesthesia and are placed intoindividual cages.

7. The animals are observed daily for signs of EAE induction (seecriteria below)

STAGE: 0 NORMAL STAGE 1 Abnormal gate and tail atony STAGE 2 Mild butdefinite weakness of one or both hind legs STAGE: 3 Severe weakness ofone or both hind legs or mild ataxia STAGE: 4 Severe paraparesis andminimal hind leg movement STAGE: 5 No hind leg movement and paraplegiaSTAGE: 6 Moribund state with no spontaneous movement and impairedrespiration. Increasing degree of front leg involvement and urinary andfecal incontinencemay also occur STAGE: 7 DEATH

Treatment is begun on day 10 after immunization. Since the diseasesymptoms in this model typically appear 10-11 days after inoculation,this time point may be considered to represent the initial phase of anacute episode of MS. It is judged that this delay of the start oftreatment mimics the clinical situation more closely than thetraditionally used protocols where drugs are administered at the timeof, or even before, inoculation (Teitelbaum D. et al., Proc Natl AcadSci USA 1999; 96: 3842-3847 and Brod S. A., et al., Ann Neurol 2000; 47:127-131).

This invention is further illustrated by the following examples ofcompounds used herein which are provided for illustration purposes andin no way limit the scope of the present invention.

SYNTHETIC EXAMPLES General

Commercial reagents and solvents were used as received. ¹H NMR spectrawere recorded on a Varian MercuryPlus-300 (300 MHz) or Varian UnityInova (400 MHz) spectrometer as indicated. Proton chemical shifts arereported in 6 ppm relative to internal tetramethylsilane (0.0 ppm). MS(LC-MS) data is obtained using a Micromass LCT time of flight massspectrometer with electrospray ionization and 5 min data acquisitiontime for m/z 100 to 1000. LC (LC-MS) is performed using a Hypersil C18column (4.6×50 mm, 3.5μ) with mobile phase of 0.1% TFA in H₂O (A) and0.1% TFA in ACN (B) and a gradient of 5% to 100% B over 3 min followedby 2 min at 100% B. Alternatively, a Platform LC-MS with electrospraysource may be used with a HP1100 LC system running at 2.0 ml/min, 200μL/min split to the ESI source with inline HP1100 DAD detection andSEDEX ELS detection. A Luna C18(2) column (30×4.6 mm 3μ) is used with agradient of 5% to 95% B over 4.5 min with mobile phase of 0.1% formicacid in H₂O and 0.1% formic acid in ACN (B). HPLC purification isperformed on a Varian ProStar system using a reversed-phase C18 columnwith a linear gradient of ACN/H₂O containing 0.1% trifluoroacetic acid.Microwave syntheses were performed using a Personal ChemistrySmithcreator microwave reaction system using 2 or 5 mL reactor vessels.

EXAMPLE 1

Intermediate:[5-Methyl-2-(4-trifluoromethyl-phenyl)-thiazol-4-yl]-aceticAcid Ethyl Ester

To a solution of 4-trifluoromethyl-benzene-thioamide (1.845 g, 9 mmol)in ethanol (15 mL, 200 proof) add ethyl-4-bromo-3-oxo-pentanoate (2.07g, 9 mmol). Seal this solution warm the solution to 170° C. in aPersonal Chemistry™ microwave oven and stir at this temperature for 20min. Cool the resulting solution to room temperature, concentrate underreduced pressure and purify the residue by flash chromatography (elutewith 30% ethyl acetate/10% dichloromethane in heptane) and obtain thetitle compound as a white solid (1.4 g). MS (ESI) m/z 330 (M+H); H1 NMR(CDCl₃) δ1.87 (bs, 1H), 2.49 (s, 3H), 4.86 (s, 2H), 7.67 (d, J=8 Hz,2H), 8.02 (d, J=8 Hz, 2H).

EXAMPLE 2

Intermediate:4-(2-hydroxy-ethyl)-5-methyl-2-(4-trifluoromethyl-phenyl)thiazole

Cool (0° C.) a solution of lithium aluminum hydride (5.3 mL, IM in THF)and add a solution of[5-methyl-2-(4-trifluoromethyl-phenyl)-thiazol-4-yl]-acetic acid ethylester (Example 1, 1.4 g, 4.25 mmol) in THF (15 mL). On completeaddition, remove the cold bath and stir for 2 hrs. Cool this solution to5° C., and then add water (0.2 mL), dropwise, followed by NaOH solution(0.2 mL, 5M in water) and water (0.2 mL). Dilute the resulting mixturewith ethyl acetate and then filter through a pad of celite. Wash thesolids with dichloromethane and then concentrate the combined filtratesunder reduced pressure. Purify the residue by flash chromatography(elute with 30% ethyl acetate 40% dichloromethane in heptane) to givethe title compound as a yellow solid (0.879 g) Use the compound ofExample 1 as the starting material to obtain the title compound.

MS (ESI) m/z 288 (M+H); H1 NMR (CDCl₃) δ2.44 (s, 3H), 2.91 (t, J=7 Hz,2H), 3.62 (t, J=6 Hz, 1H), 4.01 (dt, J=7, 6 Hz, 2H), 7.66 (d, J=8 Hz,2H), 7.96 (d, J=8 Hz, 2H). EXAMPLE 3

Intermediate:4-[5-Methyl-2-(4-trifluoromethyl-phenyl)-thiazol-4-ylethoxy]-benzoicAcid Methyl Ester

To a solution of4-(2-hydroxy-ethyl)-5-methyl-2-(4-trifluoromethyl-phenyl)thiazole(Example 3, 288 mg, 1.0 mmol) in THF (3 mL) add 4-hydroxy-benzoic acidmethyl ester (167 mg, 1.1 mmol) followed by triphenylphosphine (288 mg,1.1 mmol). To this solution, add, dropwise, diethyl azodicarboxylate(174 μL, 1.1 mmol). On complete addition, stir the resulting redsolution for 20 min. concentrate under reduced pressure and purify theresidue by flash chromatography (elute with 15% ethyl acetate/15%dichloromethane in heptane ) to give the title compound as a whitesolid. (410 mg).

MS (ESI) m/z 422 (M+H); H1 NMR (DMSO) δ2.51 (s, 3H), 3.19 (t, J=7 Hz,2H), 3.80 (s, 3H), 4.40 (t, J=7 Hz, 2H), 7.05 (d, J=9 Hz, 2H), 7.83 (d,J=8 Hz, 2H), 7.88 (d, J=8 Hz, 2H) 8.05 (d, J=8 Hz, 2H). EXAMPLE 4

Intermediate:4-{2-[5-Methyl-2-(4-trifluoromethyl-phenyl)-thiazol-4-yl]-ethoxy}-benzoicAcid Hydrazide

To a suspension of4-[5-Methyl-2-(4-trifluoromethyl-phenyl)-thiazol-4-ylethoxy]-benzoicacid methyl ester (Example 4, 410 mg, 1 mmol) in methanol (3 mL) addanhydrous hydrazine (0.32 ml, 10 mmol). Warm the resulting mixture to60° C. and stir at this temperature for 66 hrs. Cool the resultingsolution to room temperature, and add 3 drops of water. Filter theprecipitate wash with ether to give the title compound (279 mg).

MS (ESI) m/z 422 (M+H); Hi NMR (DMSO) δ2.51 (s, 3H), 3.17 (t, J=7 Hz,2H), 4.36 (t, J=7 Hz, 2H), 4.38 (bs, 2H), 6.98 (d, J=9 Hz, 2H), 7.77 (d,J=9 Hz, 2H), 7.83 (d, J=8 Hz, 2H) 8.06 (d, J=8 Hz, 2H) 9.58 (bs, 1H).EXAMPLE 5

5-(4-{2-[5-Methyl-2-(4-trifluoromethyl-phenyl)-thiazol-4-yl]-ethoxy}-phenyl)-3H-[1,3,4]oxadiazol-2-one

To a suspension of:4-{2-[5-Methyl-2-(4-trifluoromethyl-phenyl)-thiazol-4-yl]-ethoxy}-benzoicacid hydrazide (Example 4, 276 mg, 0.65 mmol) in dichloromethane (4 mL)add pyridine (104 μL, 1.3 mmol) followed by phenylchloroformate (0.88μL, 0.71 mmol). Stir the resulting mixture at room temperature until allthe starting material is consumed (by TLC analysis). Dilute the mixturewith ethyl acetate wash with water then brine dry over MgSO4 andconcentrate under reduced pressure. Take the residue up in acetonitrile(5 mL). To this mixture, add DBU (106 μL, 0.71 mmol). Seal the resultingsolution; warm it to 170° C. in a Personal Chemistry™ microwave oven andstir at this temperature for 120 min. Cool the reaction to roomtemperature, dilute with ethyl acetate, wash with 1 M HCl solution (orsaturated NaH₂PO₄ solution) dry over MgSO₄ and concentrate. Trituratethe resulting residue with dichloromethane several times to give thetitle compound as a tan solid (137 mg). (recrystallized from ethylacetate in a sealed tube at 140° C.).

MS (ESI) m/z 448 (M+H); H1 NMR (DMSO) δ2.51 (s, 3H), 3.19 (t, J=7 Hz,2H), 4.40 (t, J=7 Hz, 2H), 7.10 (d, J=8 Hz, 2H), 7.70 (d, J=8 Hz, 2H),7.83 (d, J=8 Hz, 2H) 8.05 (d, J=8 Hz, 2H) 12.41 (bs, 1H).

1 A compound of formula I:

wherein ARYL is phenyl or pyridinyl, each of which is optionally substituted with one or more substituents selected from the group consisting of halogen, C₁₋₆alkyl, C₂₋₆alkenyl, C₁₋₆alkoxy, C₁₋₆perfluoroalkyl, C₁₋₆alkylthio, hydroxy, hydroxyC₁₋₆alkyl, C₁₋₄acyloxy, nitro, cyano, C₁₋₆alkylsulfonyl, amino, C₁₋₆alkylamino and C₁₋₆alkoxycarbonyl; Z is —O(CH₂)_(n)—, —SO₂(CH₂)_(n)—, —(CH₂)_(n)—Y—(CH₂)_(n)—, —(CH₂)_(n)—CO—, —O(CH₂)_(n)—CO—, or —(CH₂)_(n)—Y—(CH₂)_(n)—CO—, wherein Y is NR₃, O or S, R₃ is selected from the group consisting of H, C₁₋₆alkyl, C₃₋₈cycloalkyl, C₁₋₆alkylC₃₋₈cycloalkyl and benzyl, and n is independently an integer from 1 to 5; X is NR₃, O or S, wherein R₃ is as defined above; R₁ is H, halogen, C₁₋₆alkyl, C₁₋₆alkoxy, C₁₋₆perfluoroalkyl; hydroxyC₁₋₆alkyl, nitro, cyano, or C₁₋₆alkylamino; and R₂ is substituted or unsubstituted phenyl, pyridinyl or thienyl wherein the substituents are selected from the group consisting of halogen, C₁₋₆alkyl, C₂₋₆alkenyl, C₁₋₆alkoxy, C₁₋₆perfluoroalkyl, C₁₋₆alkylthio, hydroxy, hydroxyC₁₋₆alkyl, C₁₋₄acyloxy, nitro, cyano, C₁₋₆alkylsulfonyl, amino, C₁₋₆alkylamino and C₁₋₆alkoxycarbonyl; provided that when Z is —O(CH₂)_(n)— or —SO₂(CH₂)_(n)—, and ARYL is phenyl, then R₂ is other than phenyl; or a stereoisomer, a tautomer or a solvate thereof, or a pharmaceutically acceptable salt thereof.
 2. The compound according to claim 1, wherein ARYL is phenyl; and X is O or S; or a stereoisomer, a tautomer or a solvate thereof, or a pharmaceutically acceptable salt thereof.
 3. The compound according to claim 2, wherein X is O; or a stereoisomer, a tautomer or solvate thereof, or a pharmaceutically acceptable salt thereof.
 4. A pharmaceutical composition comprising the compound according to claim 1, or a stereoisomer, a tautomer or a solvate thereof, or a pharmaceutically acceptable salt thereof, in combination with a pharmaceutical acceptable carrier.
 5. A method of treating a disease capable of being, modulated by PPARdelta ligand binding activity, in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a compound of formula I:

wherein ARYL is phenyl or pyridinyl, each of which is optionally substituted with one or more substituents selected from the group consisting of halogen, C₁₋₆alkyl, C₂₋₆alkenyl, C₁₋₆alkoxy, C₁₋₆perfluoroalkyl, C₁₋₆alkylthio, hydroxy, hydroxyC₁₋₆alkyl, C₁₋₄acyloxy, nitro, cyano, C₁₋₆alkylsulfonyl, amino, C₁₋₆alkylamino and C₁₋₆alkoxycarbonyl; Z is —O(CH₂)_(n)—, —SO₂(CH₂)_(n)—, —(CH₂)_(n)—Y—(CH₂)_(n)—, —(CH₂)_(n)—CO—, —O(CH₂)_(n)—CO— or —(CH₂)_(n)—Y—(CH₂)_(n)—CO—, wherein Y is NR₃, O or S, R₃ is selected from the group consisting of H, C₁₋₆alkyl, C₃₋₈cycloalkyl, C₁₋₆alkylC₃₋₈cycloalkyl and benzyl, and n is independently an integer from 1 to 5; X is NR₃, O or S, wherein R₃ is as defined above; R₁ is H, halogen, C₁₋₆alkyl, C₁₋₆alkoxy, C₁₋₆perfluoroalkyl; hydroxyC₁₋₆alkyl, nitro, cyano, or C₁₋₆alkylamino; and R₂ is substituted or unsubstituted phenyl, pyridinyl or thienyl, wherein the substituents are selected from the group consisting of halogen, C₁₋₆alkyl, C₂₋₆alkenyl, C₁₋₆alkoxy, C₁₋₆perfluoroalkyl, C₁₋₆alkylthio, hydroxy, hydroxyC₁₋₆alkyl, C₁₋₄acyloxy, nitro, cyano, C₁₋₆alkylsulfonyl, amino, C₁₋₆alkylamino and C₁₋₆alkoxycarbonyl; or a stereoisomer, a tautomer or a solvate thereof, or a pharmaceutically acceptable salt thereof.
 6. The method according to claim 5, wherein ARYL is phenyl.
 7. The method according to claim 5, wherein ARYL is phenyl; and R₂ is phenyl.
 8. The method according to claim 5, wherein ARYL is phenyl; Z is —O(CH₂)_(n)—; and R₂ is phenyl.
 9. The method according to claim 5, wherein ARYL is phenyl; Z is —O(CH₂)_(n)—; X is O or S; and R₂ is phenyl.
 10. The method according to claim 5, wherein ARYL is phenyl; Z is —O(CH₂)_(n)—; X is O or S; R₁ is C₁₋₆alkyl; and R₂ is phenyl.
 11. The method according to claim 10, wherein X is O.
 12. The method according to claim 11, wherein X is S.
 13. The method according to claim 5 wherein the disease is a demyelinating disease selected from the group consisting of multiple sclerosis, Charcot-Marie-Tooth disease, Pelizaeus-Merzbacher disease, encephalomyelitis, neuromyelitis optica, adrenoleukodystrophy, Guillian-Barre syndrome, spinal cord injuries, neuropathies, nerve injury, and disorders in which myelin forming glial cells are damaged.
 14. The method according to claim 13 wherein the demyelinating disease is multiple sclerosis.
 15. The method according to claim 5 wherein the disease is selected from the group consisting of obesity, hypertriglyceridemia, hyperlipidemia, hypoalphalipoproteinemia, hypercholesterolemia, dyslipidemia, Syndrome X, and Type II diabetes mellitus and complications thereof selected from the group consisting of neuropathy, nephropathy, retinopathy and cataracts, hyperinsulinemia, impaired glucose tolerance, insulin resistance, atherosclerosis, hypertension, coronary heart disease, peripheral vascular disease, and congestive heart failure. 